1. Technical Field
The invention relates to administering polyamide nucleic acid oligomers to living cells such that the polyamide nucleic acid oligomers engender a sequence specific biological response.
2. Background Information
Polyamide nucleic acids (PNAs) are DNA analogs containing neutral amide backbone linkages. Unlike DNA oligomers, PNA oligomers can bind DNA by displacing one strand of the duplex to form a stable D-loop structure (Peffer et al., Proc. Natl. Acad. Sci. USA 90: 10648-10652 (1993) and Mxc3x8llegaard et al., Proc. Natl. Acad. Sci. USA 91:3892-3895 (1994)). Interestingly, binding of PNA oligomers to DNA is independent of DNA strand polarity, allowing PNA oligomers to bind in both parallel and anti-parallel fashion (Egholm et al., Nature 365:566-568 (1993) and Peffer et al., Proc. Natl. Acad. Sci. USA 90:10648-10652 (1993)). In addition, PNA oligomers are less susceptible to enzymatic degradation (Demidov et al. Biochem. Pharmacol. 48:1310-1313 (1994)) and bind RNA with higher affinity than analogous DNA oligomers. Taken together, these properties suggest that PNA oligomers have great potential in both antigene and antisense approaches for regulating gene expression.
The success of an oligonucleotide analog as an antigene or antisense agent requires that the oligonucleotide be taken up by cells in reasonable quantities such that the oligonucleotide reaches its target at a sufficient concentration. PNA oligomers, however, have low phospholipid membrane permeability (Wittung et al., FEBS Letters 365:27-29 (1995)) and have been reported to be taken up by cells very poorly (Hanvey et al., Science 258:1481-1485 (1992); Nielsen et al., Bioconjugate Chem. 5:3-7 (1994); Bonham et al., Nucleic Acids Res. 23:1197-1203 (1995); Gray et al., Biochem. Pharmacol. 48:1465-1476 (1997)), which would appear to limit their potential uses in antigene and antisense approaches.
Recent strategies devised to improve cellular uptake of PNA oligomers involve conjugating other molecules to PNA sequences. Specifically, conjugating a small peptide sequence that binds the insulin-like growth factor 1 receptor (IGF1R) to a PNA oligomer increases cellular uptake of labeled PNA sequences by IGF1R-expressing cells, whereas conditions using unconjugated PNA sequences or cells lacking IGF1R show negligible cellular uptake (Basu S. and Wickstrom E., Bioconjugate Chem. 8:481-488 (1997)). These results suggest that conjugating receptor ligand molecules to PNA oligomers can increase cellular uptake; however, the ability of these receptor ligand-conjugated PNA oligomers to influence biological activity once inside the target cells remains unknown. Further, PNA oligomers will only gain entrance into cells expressing that particular targeted receptor. Thus, an appropriate ligand molecule would have to be designed and coupled to PNA oligomers for each cell type of interest. In addition, level of receptor expression can influence the permeability of ligand-conjugated PNA oligomers.
The use of PNA oligomers to manipulate brain protein expression, an approach that would greatly aid the understanding of brain function as well as neurological disease, has an additional problem. The endothelial wall of capillaries in both brain and spinal cord creates a barrier (blood-brain barrier; BBB) that excludes the uptake of molecules into these organs. Although specialized transport systems operate within the BBB to allow certain circulating molecules to cross, many pharmaceutical agents are not recognized and thus have poor BBB permeability. This appears true for PNA molecules since the transport of PNAs across the BBB is reported to be negligible (Pardridge et al., Proc. Natl. Acad. Sci. USA 92:5592-5596 (1995)). Therefore, PNA oligomers targeting brain proteins administered outside the central nervous system need to cross two barriers, the BBB and the plasma membrane of individual cells within brain, whereas PNA oligomers administered directly into brain need to cross one barrier, the plasma membrane of individual cells.
Various drug delivery strategies can circumvent the BBB permeability problem (Pardridge, Pharmacol. Toxicol. 71:3-10 (1992); Pardridge, Trends Biotechnol. 12:239-245 (1994)). For example, PNA molecules can undergo transport through the BBB when the amino terminus is biotinylated and linked to a streptavidin conjugated monoclonal antibody specific for transferrin receptor (OX26-SA; Pardridge et al., Proc. Natl. Acad. Sci. USA 92:5592-5596 (1995)). The OX26-SA antibody delivers linked molecules to brain presumably by receptor-mediated endocytosis, given the high transferrin receptor concentrations located on the BBB. These studies suggest that antibody-conjugation strategies provide a mechanism for PNA oligomers to cross the BBB. No data, however, exist as to whether the biotinylated PNA linked to OX26-SA actually enters cells or not. In addition, the utility of PNA delivery methods that rely on conjugating other molecules to PNA oligomers remains unclear since these other molecules may influence the desired functionality of particular PNAs. Therefore, strategies that enable PNA oligomers to cross biological barriers with minimal modifications of or additions to the PNA oligomer would be desirable with the ultimate goal, of course, being to influence biological activity in a sequence specific manner.
The present invention relates to PNA oligomers that influence biological activity in a sequence specific manner. Specifically, this invention relates to the discovery that PNA oligomers administered extracellularly cross biological barriers and elicit a sequence specific biological response in living cells. This discovery is in direct opposition to the current understanding of the physical properties of PNA oligomers and has far-reaching implications for both gene therapy and research purposes. Further, extracellularly administering PNA oligomers to living cells circumvents the need to micro-inject PNA oligomers directly into cells as well as the need to permeabilize cells. In addition, this invention provides for the treatment of cells in vivo such that a behavioral response is observed in an organism. Thus, this invention describes methods and materials that allow any polypeptide to be manipulated and studied in living cells. For example, the expression of a specific polypeptide can be knocked-out in adult organisms for the duration of PNA oligomer treatment. In addition to greatly aiding the advancement of basic scientific research, this ability to manipulate polypeptide expression and thus function in a sequence specific manner is clearly beneficial to gene therapy approaches involving the treatment of cancer, aging, behavioral diseases, infections, and auto-immune diseases.
One aspect of the invention provides methods of treating living cells by extracellularly administering PNA oligomers under conditions such that the PNA oligomers engender a biological response in a sequence specific manner. The PNA oligomers can be carrier-free and can be in vivo administered to an animal. The PNA oligomers are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of a living organism. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide. The polypeptides can be expressed intracranially or extracranially in a living organism. Intracranially expressed polypeptides can include those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. Specific PNA oligomers can include oligomers having sequences such as set out in SEQ ID NO:s 1 and 2. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention provides a method of screening PNA oligomers for the ability to engender a sequence specific biological response by extracellularly administering PNA oligomers to living cells. The PNA oligomers can be carrier-free and can be in vivo administered to an animal. The PNA oligomers are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of a living organism. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide. The polypeptides can be expressed intracranially or extracranially in a living organism. Intracranially expressed polypeptides can include those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention relates to a method of identifying polypeptide function by extracellularly administering PNA oligomers to living cells such that the PNA oligomers alter the expression of the polypeptide in a sequence specific manner and examining those cells for an activity that is influenced by the specific PNA oligomer. The PNA oligomers can be carrier-free and can be in vivo administered to an animal, such as a murine mammal. The PNA oligomers are capable of crossing biological barriers such as the plasma membrane of cells and the blood-brain barrier of a living organism. The PNA oligomers typically have sequence specificity for a nucleic acid sequence that encodes a polypeptide or regulates the expression of a polypeptide. The polypeptides can be expressed intracranially or extracranially in a living organism. Intracranially expressed polypeptides can include those polypeptides that participate in cell signaling. Cell signaling polypeptides include polypeptides that participate in opioid signaling. Opioid signaling polypeptides include opioid receptors, for example morphine and neurotensin receptors. Extracranially expressed polypeptides include polypeptides expressed outside the brain and cranium, for example in the gastrointestinal tract. Specific PNA oligomers can include oligomers having sequences such as set out in SEQ ID NO:s 1 and 2. The biological response engendered by the extracellular administration of PNA oligomers can be a modification, for example a reduction, of polypeptide expression. Biological responses also can be characterized by a physiological change in an animal.
Another aspect of the invention involves a method of measuring the relative turnover rate of functional polypeptides having a defined activity by extracellularly administering PNA oligomers to living cells such that the PNA oligomers influence the defined activity, and determining the time after PNA oligomer administration that the defined activity is maximally influenced. In addition, the time from when the defined activity is influenced maximally to when the activity returns to normal can be determined.
Another aspect of the invention is an article of manufacture that combines packaging material and PNA oligomers. The packaging material includes a label or package insert indicating that the PNA oligomers can be extracellularly administered to living cells for the purpose of engendering a biological response in a sequence specific manner.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.